The exception that confirms the rule: a higher-order telomeric G-quadruplex structure more stable in sodium than in potassium

DNA and RNA guanine-quadruplexes (G4s) are stabilized by several cations, in particular by potassium and sodium ions. Generally, potassium stabilizes guanine-quartet assemblies to a larger extent than sodium; in this article we report about a higher-order G4 structure more stable in sodium than in potassium. Repeats of the DNA GGGTTA telomeric motif fold into contiguous G4 units. Using three independent approaches (thermal denaturation experiments, isothermal molecular-beacon and protein-binding assays), we show that the (GGGTTA)₇GGG sequence, folding into two contiguous G4 units, exhibits an unusual feature among G4 motifs: despite a lower thermal stability, its sodium conformation is more stable than its potassium counterpart at physiological temperature. Using differential scanning calorimetry and mutated sequences, we show that this switch in the relative stability of the sodium and potassium conformations (occurring around 45°C in 100 mM cation concentration) is the result of a more favorable enthalpy change upon folding in sodium, generated by stabilizing interactions between the two G4 units in the sodium conformation. Our work demonstrates that interactions between G4 structural domains can make a higher-order structure more stable in sodium than in potassium, even though its G4 structural domains are individually more stable in potassium than in sodium.     

Amphiphilic helical peptide enhances the uptake of single-walled carbon nanotubes by living cells

The success of many projected applications of carbon nano-tubes (CNTs) to living cells, such as intracellular sensors and nanovectors, will depend on how many CNTs are taken up by cells. Here we report the enhanced uptake by HeLa cells of single-walled CNTs coated with a designed peptide termed nano-1. Atomic force microscopy showed that the dispersions were composed of individual and small bundles of nano-1 CNTs with 0.7- to 32-nm diameters and 100- to 400-nm lengths. Spectroscopic characterizations revealed that nano-1 disperses CNTs in a non-covalent fashion that preserves CNT optical properties. Elemental analyses indicated that our sample preparation protocol involving sonication and centrifugation effectively eliminated metal impurities associated with CNT manufacturing processes. We further showed that the purified CNT dispersions are taken up by HeLa cells in a time- and temperature-dependent fashion, and that they do not affect the HeLa cell growth rate, evidence that the CNTs inside cells are not toxic under these conditions. Finally, we discovered that approximately 6-fold more CNTs are taken up by cells in the presence of nano-1 compared with medium containing serum but no peptide. The fact that coating CNTs with a peptide enhances uptake offers a strategy for improving the performance of applications that require CNTs to be inside cells.     

Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins by greening cotyledons

Etiolated excised cucumber cotyledons (Cucumis sativus L. cv. Alpha Green), while greening in distilled water, synthesized and accumulated several metalloporphyrins in the absence of added substrates or inhibitors. The metalloporphyrins, undetectable by conventional spectrophotometry, exhibited distinct fluorescence emission and excitation properties in situ and in organic solvents. The metalloporphyrins were partially segregated on thin layers of silica gel H into three Chromatographic bands and the bands were eluted in methyl alcohol:acetone (4:1 $\text{v}\text{v}$). The metalloporphyrins in the eluted bands were characterized by their soret excitation and short-wavelength emission maxima. One of the metalloporphyrins of band 3 (R_f, 0.4?0.56) was identified as Mg-protoporphyrin monoester. It was accompanied by traces of two other metalloporphyrins. Band 2 (R_f, 0.32?0.48) was made up of three metalloporphyrins and had the Chromatographic mobility of endogenous protochlorophyllide. Band 1 (R_f, 0.22?0.43) was made up of two metalloporphyrins; it moved with endogenous chlorophyllide. The metalloporphyrins of band 2 and 1 exhibited fluorescence emission and excitation maxima similar to Mg-protoporphyrin monoester but slightly shifted to longer wavelengths. The Chromatographic and spectral properties of these compounds suggested that they represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The analytical techniques described in this work may prove useful in the elucidation of the enzymology between protoporphyrin IX and protochlorophyllide.     

Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability,cleavage, and cell–cell fusion

The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell–cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2–infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell–cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and transmembrane serine protease 2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell–cell fusion and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell–cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell–cell fusion. In addition, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell–cell fusion, all of which help give a more comprehensive understanding of this high-profile therapeutic target.     

Stability Studies in Biological Media of Dimer Phosphotriesters Bearing Glucuronic Acid Residue

Abstract

Hydrolytic stability of dithymidine phosphorothioates and dithioates bearing a glucuronic acid derivative protecting group on the phosphate linkage were studied in various biological media. We found that the enzymatic hydrolysis was accompanied by another reaction resulting in formation of the dithymidine phosphodiesters. We have proposed several possible mechanisms of hydrolysis.     

Qualification of Standard Membrane-Feeding Assay with Plasmodium falciparum Malaria and Potential Improvements for Future Assays

Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA). The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline Q2(R1) details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds) to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability). We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in preclinical and early clinical development of transmission-blocking vaccines, and this study strongly supports its further development and application.     

Targeting Heat Shock Protein 90 for the Treatment of Malignant Pheochromocytoma

Metastatic pheochromocytoma represents one of the major clinical challenges in the field of neuroendocrine oncology. Recent molecular characterization of pheochromocytoma suggests new treatment options with targeted therapies. In this study we investigated the 90 kDa heat shock protein (Hsp90) as a potential therapeutic target for advanced pheochromocytoma. Both the first generation, natural product Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), and the second-generation synthetic Hsp90 inhibitor STA-9090 (ganetespib) demonstrated potent inhibition of proliferation and migration of pheochromocytoma cell lines and induced degradation of key Hsp90 clients. Furthermore, ganetespib induced dose-dependent cytotoxicity in primary pheochromocytoma cells. Using metastatic models of pheochromocytoma, we demonstrate the efficacy of 17-AAG and ganetespib in reducing metastatic burden and increasing survival. Levels of Hsp70 in plasma from the xenograft studies served as a proximal biomarker of drug treatment. Our study suggests that targeting Hsp90 may benefit patients with advanced pheochromocytoma.     

Cellular and molecular processes leading to embryo formation in sponges: evidences for high conservation of processes throughout animal evolution

The emergence of multicellularity is regarded as one of the major evolutionary events of life. This transition unicellularity/pluricellularity was acquired independently several times (King 2004). The acquisition of multicellularity implies the emergence of cellular cohesion and means of communication, as well as molecular mechanisms enabling the control of morphogenesis and body plan patterning. Some of these molecular tools seem to have predated the acquisition of multicellularity while others are regarded as the acquisition of specific lineages. Morphogenesis consists in the spatial migration of cells or cell layers during embryonic development, metamorphosis, asexual reproduction, growth, and regeneration, resulting in the formation and patterning of a body. In this paper, our aim is to review what is currently known concerning basal metazoans—sponges’ morphogenesis from the tissular, cellular, and molecular points of view—and what remains to elucidate. Our review attempts to show that morphogenetic processes found in sponges are as diverse and complex as those found in other animals. In true epithelial sponges (Homoscleromorpha), as well as in others, we find similar cell/layer movements, cellular shape changes involved in major morphogenetic processes such as embryogenesis or larval metamorphosis. Thus, sponges can provide information enabling us to better understand early animal evolution at the molecular level but also at the cell/cell layer level. Indeed, comparison of molecular tools will only be of value if accompanied by functional data and expression studies during morphogenetic processes.